Method for specifically labeling living bacteria

ABSTRACT

The present invention concerns a method for labeling specifically living bacteria of a given category in a sample, the method including: a) incubating said bacteria of said sample with at least one analog of a monosaccharide compound, and b) contacting the bacteria with a labeling molecule having a second reactive group.

The present invention concerns a method for specifically labeling andmore particularly for specifically detecting living bacteria in a samplecomprising bacteria.

The traditional methods for detecting living bacteria involve theculture of the bacteria and their numbering by visualization of colonieson a solid medium. Such methods are not rapid as the culturing may takea longtime. Moreover, these methods are necessarily restricted to thedetection of cultivable bacteria and not living bacteria. Indeed, thequantification thereof is not reliable because it has been shown thatsome bacteria are killed due to the stress of the culture on solidmedium. Indeed, using plate counts, microbiologists have made oneimportant hypothesis, in a sense that they assumed that this method hadno deleterious effects on bacteria. However, and since 1950s it wasreported that apparently dead cells could be reactivated, whenscavengers (such as pyruvate, catalase, superoxide dismutase . . . ) ofreactive oxygen species, naturally produced during aerobic respiration,were added on agar plates [40-48]. For instance, various stresses likestarvation, HOCl, heat shock and desiccation, may leave cells in avulnerable physiological state in which atmospheric oxygen, during therecovery period, increases the toxic effect of the primary stressor.

In order to overcome these limitations, a number of indirectnon-culture-based methods have been proposed to assess bacterialviability, all of which have also advantages and disadvantages. Hence,there is no method that has been agreed upon as suitable in all cases.These methods assess viability by one of two criteria, demonstration ofmetabolic activity or maintenance of cellular structures.

Before the use of fluorescence technology, methods which have been usedas indications of cellular metabolic activity include the use ofmicro-autoradiography [25] as well as inducible enzyme activity [26] asan indicator of de novo protein synthesis, the direct viable countmethod (DVC) [27] which is based on the enlargement of cells uponaddition of nutrient, and the reduction of tetrazolium salts as anindication of an active electron transport chain [28]. There are,however, disadvantages associated with the use of these methods. DVC andtetrazolium salt reduction assays require nutrient addition [28, 29] andare thus dependent upon the ability of the organism to respond to thenutrient supplied. Measurements of respiration also have theirdrawbacks. A range of factors has been shown to affect formazan depositformation from tetrazolium salt reduction [29], and a report showed thatthe tetrazolium salt, 5-cyano-2,3-ditolyl tetrazolium chloride, inhibitsbacterial metabolism [30].

Cell viability assays have also been developed based on the staining ofcells with Fluorochromes. These methods assess viability by themaintenance of stable cellular structures. Acridine orange direct counts[31] and 4P, 6-diamidino-2-phenylindole staining [32] have been used asan indication of the maintenance of intact nucleic acids. Rhodamine 123has been used extensively as an indicator of membrane potential and withthe development of flow cytometry, there has been a surge of methods forcharacterization of the physiological status of the cells [33].Permeability of these dyes may be a problem in some organisms. Morerecently, some new assays have been used to assess bacterial viability,these characterize some aspect of metabolic activity (like esteraseactivity followed by the ChemChrome V6 fluorescent probe) [34], ofcellular integrity (like membrane integrity—BacLight Kit), membranepotential, intracellular pH [35] and finally ATP [36]. Finally, a newapproach has been developed since 2002 involving messenger RNA-baseddetection of viable bacterial pathogens and real-time PCR quantificationof pathogens [37].

Using methods allowing the detection of viable cell at a single celllevel, microbiologists have made one important hypothesis, in a sensethat they assume that these cells will over time be able to re-grow.However, some authors have proposed that this observation is aconsequence of cellular deterioration and that viable but non-cultivablecells are on their way to death [40, 49].

By consequence, it can be expected that the number of apparently viablecells over-estimate the number of truly alive cells (cultivable, namelyable to divide/multiply over time).

While many of these methods have the above mentioned limitations, it isfurthermore apparent that non-living bacteria maintain certaincharacteristics of viable cells which are involved in these methods,such as the enzymatic activity. Accordingly, these methods involve agreat number of false positive detection and quantification and thenumber of bacteria detected and quantified is not sufficiently reliable.

The goal of the present invention was to provide a new improved indirectnon-culture-based method for specifically detecting living bacteria,especially overcoming the above mentioned drawbacks and providing a morereliable detection and quantification of the accurate number ofcultivable bacteria.

Metabolic glycan labeling [1] has recently emerged as a very powerfultool to study cell surface glycans, with applications ranging from theirimaging in living multicellular organisms such as zebra fish or mice toidentification of metastasis associated cell-surface sialoglycoproteins[2]. This strategy relies on the assimilation by the cellularbiosynthetic machinery of a modified monosaccharide bearing a bioorthogonal chemical reporter. Metabolic incorporation of this reporterinto glycans can be further visualized by chemical ligation with a labelsuch as a fluorescent probe. Somewhat surprisingly, studies have mainlyfocused on the labeling of vertebrate glycans [3] using derivatives ofcommon monosaccharides such as N-acetyl neuraminic acid or its N-acetylmannosamine precursor, N-acetyl glucosamine, N-acetyl galactosamine andfucose.

Despite a much higher degree of diversity in their monosaccharidicbuilding blocks and an essential role in bacterium-host interactions andbacterial virulence, bacterial polysaccharides have been poorly exploredfor in vivo structural modifications. Bacteria are divided into Grampositive and Gram negative bacteria. Whereas Gram positive bacteria aresurrounded by a peptidoglycan cell wall, Gram negative bacteria arecovered by a dense layer of Lipopolysaccharides (LPS) embedded in theirouter membrane, and which are involved in the structural integrity ofthe cell and are often considered as pathogenicity determinants.

A repetitive glycan polymer contained within a LPS is referred to as theO antigen, O polysaccharide, or O side-chain of the bacteria. The Oantigen is attached to the core oligosaccharide, and comprises theoutermost domain of the LPS molecule. The composition of the O chainvaries from strain to strain. For example, there are over 160 differentO antigen structures produced by different E. coli strains. O antigen isexposed on the very outer surface of the bacterial cell. The Core domainalways contains an oligosaccharide component that attaches directly tolipid A and commonly contains sugars such as heptose and3-deoxy-D-mannooctulosonic Acid (also known as KDO, ketodeoxyoctonicacid).

Lipopolysaccharide (LPS) is one of the most important cell-surfacepolysaccharides, as it plays a key structural role in outer membraneintegrity, as well as being an important mediator of host-pathogeninteractions.

Other bacterial glycans of the outer membrane of the bacteria comprisecapsular polysaccharides (CPS), which are linear and consist ofrepeating subunits of mono-saccharides, and glycoproteins.

Glycoproteins have been shown to be important for adhesion and invasionduring bacterial infection.

Although LPS appears as an interesting target for specific andwell-defined glycan metabolic labeling in Gram negative bacteria,attempts have still been limited to the introduction of modifiedL-fucose derivatives into a purposely genetically engineered Escherichiacoli strain [4]. This last approach presents however some limitations,since:

(a) L-fucose is not generally present within the LPS of all Gramnegative bacteria, but is found in the O-antigens of specific strains[5],

(b) Free L-fucose is not an intermediate in the normal E. coli “de novo”pathway, and should therefore not be directly activatable into anucleotide-sugar donor [6] without the introduction by geneticengineering of an alternative pathway, known as “salvage pathway”, intothe organism of interest (metabolic pathway engineering), and

(c) Once activated in the form of GDP-Fuc, the modified L-fucoseanalogue might, following the reverse de novo pathway, be transformedinto GDP-Man and potentially further metabolized into various othercompounds, the chemical reporter being now susceptible to spread throughother pathways of the sugar metabolism (or beyond).

As the above mentioned LPS labeling on bacteria required thereforegenetic modification of the bacteria, it could not be used in a methodof detecting any living bacteria in a tested sample according to thepresent invention.

According to the present invention it has been investigated whetherother sugars could be used as a target for glycan metabolic modificationof at least a given category of bacteria without requiring geneticmodification of the said bacteria, taking advantage of the fact thatmetabolic modification would then be an evidence of viability of thebacteria.

The method of the present invention therefore comprises essentiallydetecting viable bacteria in labeling the bacterial membranes thereofvia metabolic modification of membrane glycans thereof, especially theLipopolysaccharides, by incorporating into their membrane glycans,especially their LPS, a modified sugar component bearing a bioorthogonal chemical reporter, thus decorating the cell surface andenabling the detection thereof with a chemistry reaction, especiallyclick chemistry reaction, allowing further detecting of the chemicalreporter, in an overall rapid process as schematically shown on FIG. 2B.

According to the present invention it has been found that modifiedsugars comprising ulosonic acid or ulosonate residue are particularlyadvantageous in that such residues can be found in glycans of thebacterial membrane, especially LPS of all of the Gram negative bacteria,and moreover they can be directly assimilated in the form into whichthey will be incorporated in the said glycans, especially the LPS ofGram negative bacteria, while most of the other sugars of the LPS comefrom a different monosaccharidic precursor.

Ulosonic acids (also called ketoaldonic acids, or aldulosonic acids) aremonosaccharides of the ketose family, presenting a ketone function atC-2, and a carboxylic acid at C-1. Octulosonic and nonulosonic acids arefound in diverse natural glycans, including different forms of bacterialglycans (especially LPS, CPS, glycoproteins). The biosynthetic pathwayleading to the elaboration of these glycans generally involves the freeulosonic acid as an intermediate, which is then directly activated inthe form of a CMP-sugar donor. This is in contrast with many othermonosaccharides which are often biosynthesized directly in the form ofnucleotide diphosphate-sugar donors from a small set of primaryactivated donors of simple monosaccharides.

The present invention provides a method for labeling specifically livingbacteria of a given category of bacteria in a sample comprisingbacteria, in incorporating a first reactive chemical group bound to theglycans of the outer membrane of said living bacteria.

More accurately, the present invention provides a method forspecifically labeling living bacteria of a given category of bacteria ina sample comprising bacteria, the method comprising the steps of:

a) incubating said bacteria of said sample with at least one analog of amonosaccharide compound, said monosaccharide being an endogenousmonosaccharide residue of glycans of the outer membrane of such givencategory of bacteria, the said endogenous monosaccharide residuecomprising an ulosonic acid or ulosonate salt residue, the said analogof a monosaccharide compound being a modified monosaccharide substitutedat a given position by a first reactive chemical group capable to reactwith a second reactive group of a labeling molecule, the said givenposition being preferably a position which comprises a free group in thesaid endogenous monosaccharide residue incorporated within said glycansof the outer membrane of the bacteria,

b) contacting said bacteria with a said labeling molecule comprising asaid second reactive group, for generating the reaction of said firstreactive group of said analog residue incorporated within said glycansof the outer membrane of said living bacteria with said second reactivegroup of said labeling molecule.

Living bacteria comprise bacteria capable of multiplying as well asviable bacteria not capable to multiply. As most of the sanitaryregulations refer to the numbering of bacteria capable to multiply,especially capable to multiply on a solid growth medium, advantageously,the present invention provides more particularly a method for labelingspecifically bacteria capable of multiplying wherein said bacteria areincubated in a culture medium in (liquid medium) or on (solid medium)which said bacteria are capable to multiply.

Preferably, the method of the present invention comprises the furtherstep of:

c) detecting living bacteria in detecting whether said bacteria compriselabeling molecule bound to the glycans of their outer membrane and/orimmobilizing living bacteria bearing said labeling molecule, onto asolid substrate.

The said detecting step c) can be carried out in a liquid medium or on asolid substrate.

More particularly, said labeling molecule is a detectable moleculecomprising a detectable substance or capable to react or to be bound toa detectable substance or said labeling molecule is a first moleculebearing a said second reactive group, said first molecule being capableto react or to be bound to a second molecule and/or to a solidsubstrate, preferably said second molecule comprising a detectablesubstance and/or said second molecule being bound to a said solidsubstrate.

In a first embodiment, said labeling molecule can be a detectablemolecule, namely a molecule consisting in or bearing a detectablesubstance, namely a substance capable to be detected such as afluorochrome or luminescent substance or an enzyme such as peroxidase,said enzyme being more particularly detected after reacting with acoreactant.

In a further particular embodiment, useful for isolating livingbacteria, the said labeling molecule can be bound to a solid substratewhen carrying out step b).

In a further particular embodiment, said labeling molecule is moleculewhich is a first ligand or first binding protein bearing a said secondreactive group and in step c) said living bacteria coupled to said firstligand or first binding protein is detected and/or immobilized bycontacting said first ligand or first binding protein with a secondmolecule which is a second ligand or second binding protein reacting orbinding specifically to said first ligand or first binding protein.

Then, advantageously, said first or second ligand or binding protein canreact or be bound to a third binding protein bearing a said detectablesubstance such as a fluorochrome or luminescent substance or an enzymesuch as peroxidase, said third binding protein binding specifically to asaid first and/or second ligand or binding protein. Detecting saiddetectable substance via a said second ligand or second binding proteinor third binding protein enables to amplify the signal of the saiddetectable substance.

More particularly, the first ligand or first binding protein can be:

-   -   biotin, said second and third binding protein being then avidin        or streptavidin and, respectively, an antibody raised against        biotin, or    -   avidin or streptavidin, said second ligand and said third        binding protein being then biotin and, respectively, an antibody        raised against avidin or streptavidin, or    -   a first antibody, said second and third binding protein being        then a second and third antibody specific to said first        antibody.

More particularly, said labeling molecule is a first ligand, preferablybiotin, bearing a said second reactive group, and in step c) said livingbacteria coupled to said first ligand are detected by reaction of saidbacteria with an antibody specific to said first ligand, said antibodybearing a detectable substance, preferably a fluorochrome or luminescentmolecule or an enzyme.

More particularly again, said labeling molecule is a first ligand,preferably biotin, bearing a said second reactive group, and in step c)said living bacteria coupled to said first binding protein isimmobilized by reacting said first ligand with a solid substrate,preferably magnetic beads, coupled to a said second binding protein,preferably avidin or streptavidin, before detecting said living bacteriaby bacterial DNA enzymatic amplification or by reaction of said bacteriawith a third binding protein reacting or binding specifically to saidfirst ligand or second binding protein, said third binding proteinbearing a detectable substance, preferably a fluorochrome or luminescentmolecule or an enzyme, said third binding protein being preferably anantibody specific to said first ligand or first binding protein.

Such embodiment wherein said living bacteria are immobilized on saidsolid substrate enables to concentrate the sample into said bacteria andto quantify said living bacteria by any known method including DNAenzymatic amplification such as PCR, especially Real Time PCR or amethod involving immunological reaction with a labeled antibody such asan ELISA test.

More particularly, the present invention provides a method forspecifically detecting living bacteria of a given category of bacteriain a sample comprising bacteria, said labeling molecule being adetectable molecule comprising a detectable substance, the methodcomprising the step c) of detecting living bacteria in detecting whethersaid bacteria comprise said detectable molecule bound to the glycans oftheir outer membrane.

Step a) results in labeling specifically all living bacteria of saidcategory by incorporating into the glycans, especiallylipopolysaccharide layer of the outer membrane of the living bacteria, aresidue of said analog of monosaccharide compound.

Step b) results in bonding the said labeling molecule to the analogresidue incorporated to the said glycans of living bacteria, especiallyLPS, by reacting the said first reactive group of the said modifiedmonosaccharide within the said glycans, especially LPS of saidcultivated bacteria, with a said labeling molecule comprising a saidsecond reactive group under appropriate conditions and in the presenceof appropriate suitable reactants.

In step a), preferably the said given position of the said endogenousmonosaccharides is a position which comprises a free group in the saidendogenous monosaccharide residue incorporated within said glycans ofthe outer membrane of the bacteria. By “free group” is meant a positionnot engaged in a covalent bond within the said glycans, especially LPS.

The modified ulosonic acid or ulosonate salt being metabolicallyassimilated led to the use of this method for fast detection—the overallprocess taking less than one day—of metabolically active/viable Gramnegative bacteria. This method is very powerful in regard to the factthat detection of viable bacteria needs normally between 2 days and morethan one month depending on the bacterial strain.

Severe pathogens are hiding amongst Gram negative bacteria, and therapid identification of viable cells represents a major sanitarychallenge. The present invention therefore provides a simple strategy tolabel the cell surface of metabolically active Gram negative bacteria,via metabolic incorporation of a modified ulosonic acid or ulosonateresidue into their lipopolysaccharide, followed by further conjugationusing click-chemistry.

The assimilation of an exogenous ulosonate analog, such as a KDO analog,in the bacterial cell, and its incorporation into the LPS in competitionwith its endogenous counterpart, such as KDO, was not obvious at all,since these molecules are highly polar and do not cross membraneseasily. Attempts at using KDO analogs as antibacterials have failed dueto the difficulty to reach a sufficient intracellular concentration ofthese analogs [10].

More particularly, the incubation time at step a) is from 1 hr to 24 hr,preferably from 2 hr to 12 hr and the monosaccharide analogue compoundconcentration is from 10⁻⁸M to 1M, more particularly 10⁻⁵M to 1M, fordetecting a bacteria concentration of no more than 10¹¹ cell/ml, moreparticularly no more than 10⁹ cell/ml.

Particularly, the said analog monosaccharide is a substituted ulosonicacid having one of the following formula (I) or (II) or an ulosonatesalt thereof:

Wherein

-   -   A, B and C can be independently H, OH, NH₂, OH and NH₂ being        substituted or not by protecting groups thereof, preferably OH        and NH₂ being substituted by alkyl, hydroxyalkyl, acyl, formyl        or imidoyl groups, and    -   D is an alkyl chain in C₂ to C₄, each carbon being substituted        or not by OH or NH₂, OH and NH2 being substituted or not by        protecting groups thereof, preferably OH and NH₂ being        substituted by alkyl, hydroxyalkyl, acyl, formyl or imidoyl        groups, and    -   at least one of A, B, C or D groups is substituted by a said        first reactive group.

These two formula one with a pyranosic cycle (I) and the other with afuranosic cycle (II) correspond in fact to the two species naturally inequilibrium although generally the formula (I) is predominant.

More particularly, for OH the protecting group can be preferably analkyl, hydroxyalkyl, acyl or formyl group.

More particularly, for NH2 the protecting groups can be selected amongalkyl, hydroxyalkyl, acyl, formyl or imidoyl groups.

NH2 can be protected by one or two protecting groups, especially one CH3group and one alkyl, hydroxyalkyl, acyl, formyl or imidoyl group,especially acetyl (Ac), acetimidoyl (Am), N-methyl-acetimidoyl,N,N-dimethyl-acetimidoyl, formyl (Fo), or hydroxybutanoyl group.

More particularly, the said analog of monosaccharide compound is asubstituted octulosonic acid or octulosonate salt compound.

An octulosonic or octulosonate compound of formula (I) comprises as Dgroup an alkyl chain in C₂.

An octulosonic acid or octulosonate compound of formula (II) comprisesas D group an alkyl chain in C₃.

The said analog of monosaccharide compound can also be a substitutednonulosonic or nonulosonate salt compound.

A nonulosonic or nonulosonate compound of formula (I) comprises as Dgroup an alkyl chain in C₃.

A nonulosonic or nonulosonate compound of formula (II) comprises as Dgroup an alkyl chain in C₄.

Among the endogenous ulosonic acid or ulosonate residues, particularlyfrequent are the following ones which are specifically present in thefollowing categories of bacteria:

1—Octulosonic acid or octulosonate residues of the following formula (I)wherein D is —CHOH—CH₂OH (Ia-1, Ia-2) or —CHOH—CH₂NH₂ (Ia-3):

(3-deoxy-D-manno-oct-2-ulosonic acid, or ketodeoxyoctonic acid),specific to the following category of bacteria: all Gram negativebacteria. It can be found in the LPS inner core of all Gram negativebacteria except some Shewenella bacteria, as well as the LPS O-antigenof Providencia, Cronobacter and Pseudoalteromonas bacteria and alsofound in some capsular polysaccharides (CPS) in E. coli, Neisseriameningitidis, rhizobia, Actinobacillus pleuropneumoniae, Moraxellanonliquefaciens, Burkholderia pseudomallei, Burkholderia cepacia,Burkholderia caribensis, Pseudoalteromonas nigrifaciens).

(D-glycero-D-talo-oct-2-ulosonic acid, or ketooctonic acid), (Ia-2) canbe found in to the following category of bacteria: Yersinia,Acinetobacter, Burkholderias.

(8-amino-3,8-dideoxy-D-manno oct-2-ulosonic acid, (Ia-3) can be found inthe Schewanella bacteria:

2—Nonulosonic acid or nonulosonate residues of the following formulawherein D is —CHNH₂—CHOH—CH₃ (Ib-1, Ib-2, Ib-5, Ib-6), —CHOH—CHOH—CH₂OH(Ib-3,Ib-4), —CHNH₂—CHNH₂—CH₃ (Ib-7):

(5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid),(Ib-1) can be found in the LPS of Legionella pneumophila bacteria and inSchewanella japonica.

(5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonicacid), (Ib-2) can be found in E. coli strains, Providencia stuartii,Pseudomonas aeruginosa, Yersinia ruckeri, Salmonella arizonae,Morganella morganii, Shewanella putrefaciens.

(3-Deoxy-D-glycero-D-galacto-non-2-ulosonic acid), (Ib-3) can be foundin the CPS of Klebsiella ozaenae and Sinorhizobium fredii (in the formof 5-O-methyl Kdn). Kdn-containing polysaccharides or oligosaccharideshave been found in the cell wall of Gram positive bacteria (order ofActinomycetales).

(5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid), (Ib-4)can be found in the CPS of E. coli, Neisseria meningitidis, Moraxellanonliquefaciens, and Mannheimia (Pasteurella) haemolytica, Streptococcusagalactiae (Gram+), Streptococcus suis (Gram +) and in the LPS O-antigenof bacteria including Hafnia alvei, Escherichia albertii, Salmonellaenterica, E. coli, Citro-bacter, Vibrio cholerae, Shewanella algae, andin the LPS core of several pathogens including N. meningitidis,Neisseria gonorrhoeae, H. influenzae, Haemophilus ducreyi, Histophilussomni, Campylobacter jejuni, and Helicobacter pylori.

(5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid)(Ib-5) can be found in the O-antigen (LPS) of Pseudomonas aeruginosa,Shigella boydii, Escherichia coli, Proteus vulgaris, Pseudoalteromonasatlantica, Pseudoalteromonas distincta, Sinorhizobium fredii, and Vibriocholerae, Pseudoalteromonas atlantica and Cell wall of Kribella spp. 5(Gram +) and Actinoplanes utahensis (Gram +) and LPS core of Vibrioparahaemolyticus and in flagellar glycoproteins of Gram positiveCampylobacter jejuni, Campylobacter coli, Helicobacter pylori, andClostridium botulinum, and in the CPS of Sinorhizobium bacteria.

(5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonicacid), (Ib-6). can be found in Legionella pneumophila, Vibrioalginolyticus, Acinetobacter baumannii, Pseudomonas fluorescens, andVibrio salmonicida.

-   -   (Ib-7):5,7,8-triamino-3,5,7,8,9-pentadeoxynon-2-ulosonic acid        (unknown configuration at C-8) can be found in Tenacibaculum        maritimum (former Flexibacter maritimus).

In the above formula Ib-i, with i=1 to 7, NH₂ groups can be in the formof N-acetyl (NHAc), or can be in the form of N-acetimidoyl (NHAm),N—(N-methylacetimidoyl), N—(N,N-dimethylacetimidoyl), N-formyl (NHFo),NH-hydroxybutanoyl (NH-Hb), and can be further N-methylated or not.

Preferably, the said given category of bacteria comprises Gram negativebacteria and said endogenous monosaccharide residue of said LPS layer ofthe outer membrane of the bacteria is a deoxyoctulosonate residue andsaid analog of monosaccharide compound is a substituted deoxyoctulosonicacid or deoxyoctulosonate compound (also named ketodeoxyoctonate,so-called KDO).

By “Gram negative bacteria”, it is meant that the method with the saidKDO does not allow labeling of Gram positive bacteria and other Gramnegative bacteria not bearing the same endogenous monosaccharide.Indeed, all of the Gram negative bacteria LPS comprise a saiddeoxyoctulosonate residues while it is not comprised in the glycans ofGram positive bacteria.

By “deoxyoctulosonate” also referred to as Kdo is meant a3-deoxy-D-manno octulosonic acid salt residue of formula (Ia-1).

More particularly, the said deoxyoctulosonate residue is substituted bya said reactive group at a position selected among the positions 3, 4,5, 7 or 8 of the monosaccharide cycle, preferably 3, 7 or 8. Theposition 2 of this endogenous monosaccharide is engaged in a covalentbond with the LPS.

All of the Gram negative bacteria comprise a said deoxyoctulosonateresidue comprising free groups which can be substituted by a said firstreactive group at one of these positions 3, 4, 5, 7 or 8 of at least oneresidue and all of the Gram negative bacteria comprise a free group atthe position 8 of at least one residue except some Shewenella bacteria.

To detect specifically the Gram negative bacteria, it can be moreadvantageous to use a culture medium specific to Gram negative bacteriain steps a) and b) therefore not allowing culture of Gram positivebacteria.

More particularly, the said deoxyoctulosonate residue is a compound offormula (I) or (II), more particularly of formula (Ia-1), substituted bya said reactive group R₁ at the position 8 wherein D=—CHOH—CH₂—R₁, A=H,B=OH, C=OH in formula (I) or D=—CHOH—CHOH—CH₂—R₁, A=H, B=OH in formula(II).

More particularly, the said Gram negative bacteria having an endogenousincorporated KDO are selected among E. coli, Salmonella typhimurium,Legionella pneumophila and Pseudomonas aeruginosa. These bacteriapresent endogenous KDO free at the position 8.

Other Gram negative pathogens bacteria having at least one the positionsof an ulosonic acid or ulosonate residue free can be selected amongBacteroides fragilis, Bartonella bacilliformis, Bartonella quintana(Rochalimaea quintana), Bartonella spp. (Rochalimaea spp.), Bordetellabronchiseptica, Bordetella parapertussis, Bordetella pertussis,Brachyspira spp, Campylobacter fetus, Campylobacter jejuni,Campylobacter spp, Cardiobacterium hominis, Chlamydophila abortus,Chlamydophila caviae, Chlamydophila felis, Chlamydophila pneumoniae(Chlamydia pneumoniae), Edwardsiella tarda, Ehrlichia spp, Eikenellacorrodens, Elizabethkingia meningoseptica (Flavobacteriummeningosepticum, Chryseobacterium, eningosepticum), Enterobacteraerogenes (=Klebsiella mobilis), Enterobacter cloacae, Enterobacter spp,Enterococcus spp, Francisella tularensis subsp. holarctica (“Francisellatularensis var. palaearctica”), Francisella tularensis type B),Fusobacterium necrophorum, Haemophilus ducreyi, Haemophilus influenzae,Haemophilus spp, Helicobacter pylori, Campylobacter pylori, Klebsiellaoxytoca, Klebsiella pneumoniae, Klebsiella spp, Legionella bozemanaecorrig. (Fluoribacter bozemanae), Legionella pneumophila, Legionellaspp, Leptospira interrogans, Leptospira interrogans sensu lato inclutLeptospira alexanderi, Leptospira borgpetersenii, Leptospira fainei,Leptospira inadai, Leptospira interrogans, Leptospira kirschneri,Leptospira noguchii, Leptospira santarosai, Leptospira weilii,Morganella morganii (Proteus morganii), Neisseria gonorrhoeae, Neisseriameningitidis, Neorickettsia sennetsu (Ehrlichia sennetsu, Rickettsiasennetsu), Pasteurella multocida, Pasteurella spp, Plesiomonasshigelloides, Porphyromonas spp, Prevotella spp, Proteus mirabilis,Proteus penneri, Proteus vulgaris, Providencia alcalifaciens,Providencia rettgeri (Proteus rettgeri), Providencia spp, Pseudomonasaeruginosa, Rickettsia spp, excluding Orientia (Rickettsia)tsutsugamushi, Rickettsia akari, Rickettsia canadensis, Rickettsiaconorii, Rickettsia montanensis, Rickettsia prowazekii, Rickettsiarickettsia et Rickettsia typhi, Salmonella enterica subsp. Arizonae(Salmonella arizonae, Salmonella choleraesuis subsp. arizonae),Salmonella enterica subsp. enterica sérovar Enteritidis (Salmonellaenteritidis), Salmonella enterica subsp. enterica sérovar Paratyphi A(Salmonella paratyphi), Paratyphi B, and Paratyphi C, Salmonellaenterica subsp. enterica sérovar Typhimurium (Salmonella typhimurium),Shigella boydii, Shigella dysenteriae, except type 1, Shigella flexneri,Shigella sonnei, Streptobacillus moniliformis, “Treponema carateum,Treponema pallidum, “Treponema pertenue” (“Treponema pallidum subsp.pertenue”), Treponema spp, Vibrio cholerae, vibrio parahaemolyticus(=Beneckea parahaemolytica), Vibrio spp, Yersinia enterocolitica,Yersinia pseudotuberculosis, Brucella melitensis (sensu stricto),Brucella melitensis biovar Abortus (Brucella abortus), Brucellamelitensis biovar Canis (Brucella canis), Brucella melitensis biovarSuis (Brucella suis), Burkholderia mallei (Pseudomonas mallei),Burkholderia pseudomallei (Pseudomonas pseudomallei), Chlamydophilapsittaci (Chlamydia psittaci), Coxiella burnetii, Francisella tularensissubsp. Tularensis (“Francisella tularensis subsp. nearctica”,Francisella tularensis biovar Tularensis, Francisella tularensis typeA), Orientia tsutsugamushi (Rickettsia tsutsugamushi), Rickettsia akari,Rickettsia canadensis corrig, Rickettsia conorii, Rickettsia montanensiscorrig, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi,Salmonella enterica subsp. enterica sérovar Typhi (Salmonella typhi,Shigella dysenteriae type 1, Yersinia pestis.

In another preferred embodiment, the method according to the inventionis for labeling, preferably detecting, specifically living Legionellapneumophila bacteria and the said given category of bacteria is thecategory of the Legionella pneumophila bacteria and said endogenousmonosaccharide residue of said LPS layer of the outer membrane of thebacteria is a 4-epilegionaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid) or4-epilegionaminate residue, or a legionaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid)or legionaminate residue, and the said analog of a monosaccharidecompound is respectively a substituted 4-epilegionaminic acid or4-epilegionaminate compound, or a substituted legionaminic acid orlegionaminate compound, preferably substituted at one position selectedamong the positions 3, 4, 5, 7, 8 and 9 of the monosaccharide cycle,preferably 5, 7 and 9. The position 2 of this endogenous monosaccharideis engaged in a covalent bond with the LPS.

Preferably, the said analog of a monosaccharide compound is asubstituted 4-epilegionaminic acid or 4-epilegionaminate compound offormula (I), more particularly (Ib-1), or a substituted legionaminicacid or legionaminate compound of formula (I), more particularly (Ib-6),substituted by a said reactive group R₁ at the position 9 whereinD=—CHNH₂—CHOH—CH₂R₁, A=H, B=OH and C=NH₂.

Another preferred structure of the said analog of a monosaccharidecompound is a substituted 4-epilegionaminic acid or 4-epilegionaminatecompound of formula (I), more particularly (Ib-1), or a substitutedlegionaminic acid or legionaminate compound of formula (I), moreparticularly (Ib-6), substituted by a said reactive group R₁ at, atleast, one of the positions 5 and 7, wherein D=—CHNHR₂—CHOH—CH₃, A=H,B=OH and C=NHR₃, with R₂ and R₃ being independently one of each othereither H or R₁.

In another preferred embodiment, the method according to the inventionis for labeling, preferably detecting, specifically living Pseudomonasaeruginosa bacteria and the said given category of bacteria is thecategory of the Pseudomonas aeruginosa bacteria and said endogenousmonosaccharide residue of said LPS layer of the outer membrane of thebacteria is a 8-epilegionaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid)or 8-epilegionaminate residue, or a pseudaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid)or pseudaminate residue, and the said analog of a monosaccharidecompound is respectively a substituted 8-epilegionaminic acid or8-epilegionaminate compound, or a substituted pseudaminic acid orpseudaminate compound, preferably substituted at one position selectedamong the positions 3, 4, 5, 7, 8 and 9 of the monosaccharide cycle,preferably 5, 7 and 9. The position 2 of this endogenous monosaccharideis engaged in a covalent bond with the LPS.

Preferably, the said analog of a monosaccharide compound is asubstituted 8-epilegionaminic acid or 8-epilegionaminate compound offormula (I), more particularly (Ib-2), or a substituted pseudaminic acidor pseudaminate compound of formula (I), more particularly (Ib-5),substituted by a said reactive group R₁ at the position 9 whereinD=−CHNH₂—CHOH—CH₂R₁, A=H, B=OH and C=NH₂.

Another preferred structure of the said analog of a monosaccharidecompound is a substituted 8-epilegionaminic acid or 8-epilegionaminatecompound of formula (I), more particularly (Ib-2), or a substitutedpseudaminic acid or pseudaminate compound of formula (I), moreparticularly (Ib-5), substituted by a said reactive group R₁ at, atleast, one of the positions 5 and 7, wherein D=—CHNHR₂—CHOH—CH₃, A=H,B=OH and C=NHR₃, with R₂ and R₃ being independently one of each othereither H or R₁.

More particularly, the said detectable substance is a fluorochrome orluminescent molecule detectable by fluorescence or luminescence.

Preferably, the said first reactive group R₁ is selected among groupsconsisting in or bearing the group azido (—N₃) and groups consisting inor bearing the group alkyne (—C≡C—), and the said second reactive groupR₂ is selected among groups consisting in or bearing respectively thegroups alkyne (—C≡C—) and azido (—N₃), and reacting the said azidoreactive group with a said alkyne group (—C≡CH), is carried out inperforming an azide alkyne cycloaddition.

An azide alkyne cycloaddition is a well-known so-called click chemistryreaction in the presence of a Copper catalyst wherein the azide groupreacts with the alkyne group to afford a triazole.

More particularly, the reaction is carried out in copper catalyzedconditions in the presence of a tris-triazolyl ligand, preferably TGTA.

More particularly, the detectable molecule is a fluorophore bearing aterminal alkyne group.

More particularly, the Kdo analog replacing endogenous 3 deoxy-D-mannooctulosonic acid incorporated into the LPS layer of the outer membraneof the bacteria, is a Kdo-N₃ (8-azido-3,8-dideoxy-D-manno-octulosonicacid) analog reacting in the presence of a tris-triazole ligand such asTGTA (Tris((1-(β-D-glucopyranosyl)-1H-[1,2,3]-triazol-4-yl)methyl)amine)or TBTA (Tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) and anAlexa labeling fluorophore molecule bearing a terminal alkyne group witha catalyst so as to perform an azide alkyne cycloaddition of the saidfluorophore and said Kdo-N₃.

Other appropriate ligands frequently used are:tris(3-hydroxypropyltriazolylmethyl)amine (THPTA),2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethanesulfonicacid (BTTES), Tris((1-((O-ethyl) carboxymethyl)-(1,2,3-triazol-4-yl))methyl)amine, Bathophenanthroline disulfonate, orTris(2-benzimidazolylmethyl)amines (53).

Other reactive groups and other reactions are possible such as:Staudinger Ligation (first reactive group=azide and second reactivegroup=phosphine), copper-free click-chemistry (first reactivegroup=azide and second reactive group=constrained alkyne (intracyclicalkyne)), carbonyl condensation (first reactive group=aldehyde or ketoneand second reactive group=hydrazide or oxyamine), His-tag (firstreactive group=oligo-histidine and second reactive group=nickel-complexor nickel ligand), thiol-ene click chemistry (first reactive group=thioland second reactive group=alkene), nitrile-oxide-ene click chemistry(first reactive group=nitrile oxide or aldehyde, oxime, or hydroxymoylchloride or chlororoxime and second reactive group=alkene or alkyne),nitrile imine-ene click chemistry (first reactive group=nitrile imine oraldehyde, hydrazone, or hydrazonoyl chloride or chlorohydrazone andsecond reactive group=alkene or alkyne), inverse electron demandDiels-Alder ligation (first reactive group=alkene and second reactivegroup=tetrazine), Suzuki-Miyaura coupling (first reactive group=arylhalide and second reactive group=aryl boronate).

In the above-mentioned listing of groups involved in the reactions, thefirst reactive group and the second reactive group can be permuted.

Other and higher specificity of detection can be obtained in incubatingthe bacteria sample with two said different monosaccharide analogcompounds and two different detectable molecules.

In another particular embodiment of the method of the present invention,the said incubation of step a) and reaction of step b) are carried outon a membrane filter so that the cultivated bacteria emanating from asame original bacterium which has been multiplied are grouped togetherand can be visualized with a microscope and the said detectable moleculecan be detected by visualization with a said microscope. Therefore, thenumber of cultivable bacteria can be quantified thereby.

This embodiment enables to filter the tested sample on said membranefilter such as a polyester membrane, prior to assimilation of the saidmodified monosaccharide to avoid over-estimation of viable bacteria dueto possible growth during the assimilation period. Indeed, when cellsfixed on the top of such membrane start to grow, they stay together andform a micro colony that can be easily detected as coming from the samesingle cell. Therefore, this enables to number by counting thecultivable bacteria.

The present invention also provides a kit for carrying out the method ofthe invention comprising:

-   -   a said analog of a monosaccharide compound comprising an        ulosonic acid or ulosonate compound substituted at a given        position by a said first reactive chemical group, and    -   a said labeling molecule comprising a said second reactive group        capable of reacting with said first reactive group, and    -   reactants for generating the reaction of said first reactive        group of said analog residue incorporated within said glycans of        the outer membrane of said bacteria with said second reactive        group of said labeling molecule, and    -   a culture or incubation medium allowing the growth of a said        given category of bacteria, preferably specific to the growth of        said given category of bacteria, and

Preferably, the said culture or incubation medium further comprisesagents enhancing and/or accelerating the growth speed and/or thecapacity to form colonies of the said given category of bacteria. Moreparticularly, the incubation medium comprises at least an antioxidantagent such as pyruvate or catalase.

More particularly, in one embodiment, the kit further comprises:

-   -   a said detectable molecule or said second molecule bearing a        detectable substance, preferably a fluorochrome or luminescent        molecule or an enzyme, and/or    -   a solid substrate bearing a said second molecule capable of        specifically reacting or binding with said labeling molecule.

More particularly, in one embodiment, the kit of the present inventionfurther comprises:

-   -   a said detectable molecule comprising a said second reactive        group capable of reacting with said first reactive group, and    -   a solid medium allowing visualization of the bacteria after        incubating with the said analog of a monosaccharide compound,        said reactants and said detectable molecule.

More particularly again, the kit comprises:

-   -   a said analog of a monosaccharide compound being a        deoxyoctulosonic acid or deoxyoctulosonate salt compound        substituted by a said first reactive group comprising an azido        or alkyne group, and    -   a said second reactive group of the detectable molecule bearing        an alkyne or, respectively, azido group, and    -   said reactants comprising a copper catalyst and a tris-triazolyl        ligand.

Other characteristics and advantages of the present invention will bemore apparent in the light of the following detailed descriptionreferring to the following figures wherein:

FIG. 1A shows the Structure of the major component of E. coli K12lipopolysaccharide, with the KDO sugar residue incorporated within IC,the Inner core of the LPS of Gram negative bacteria between OC, theouter core and LA, the lipid A, showing the anchoring site A forO-antigen and the possible site of modification B of KDO in position 8;Glc: D-glucose; GlcN: 2-amino-2-deoxy-D-glucose; KDO:3-deoxy-D-manno-octulosonate; Hep: L-glycero-D-manno-heptose; Gal:D-galactose;

FIG. 1B shows the KDO biosynthetic pathway (KDO pathway);

FIG. 2A shows the molecules used in this example (1=KDO-N₃, 2=TGTA,3=A488-yne);

FIG. 2B shows a schematic representation of metabolic LPS labelling ofE. coli with 1=KDO-N₃;

FIG. 3A represents photography of E. coli K12 with metabolicallyincorporated KDO-N₃ (+KDO-N₃) and without metabolically incorporated KDO(−KDO-N₃) before it is revealed via A488-yne—Cu(I) catalyzed clickchemistry after 5 min. (in grey) and after it is revealed (in black);

FIG. 3B represents a graphic of frequency distribution of the bacterialfluorescence values generated and plotted with (grey bars) or without(black bars) adding KDO-N₃, the arbitrary unit of fluorescence(“a.u.f.”) range being from 0 to 1300 in abscissa and the relativefrequency (“r.f.”) range being from 0 to 1;

FIG. 3C is a photography showing fluorescence concentrated at thecellular surface of E. coli labelled via A488-yne—Cu (I) catalyzed clickchemistry during 5 min, the image being deconvolved usingRichardson-Lucy algorithm with an experimental point spread fusion;

FIG. 4 are photographs representing the detection of metabolicallyincorporated KDO-N₃ by various bacterial strains, metabolicallyincorporated KDO-N₃ by various bacteria was revealed via A488-yne—Cu(I)catalyzed click chemistry after 60 min. Phase contrast and fluorescenceimages (Left panel) without adding KDO-N₃; (right panel) with addingKDO-N₃. Scale bar 1 μm as in FIG. 3 A (bacteria with added KDO-N₃(+KDO-N₃) and without added KDO (−KDO-N₃) before it is revealed viaA488-yne—Cu(I) catalyzed click chemistry after 5 min in grey and afterit is revealed in black; and

FIG. 5 represents the results (photographs and graphics) of detection ofmetabolically incorporated KDO-N₃ by various bacterial strains.Metabolically incorporated KDO-N₃ by various bacterial strains wasrevealed via A488-yne—Cu(I) catalyzed click chemistry after 60 min.Frequency distribution of the bacterial fluorescence values wasgenerated and plotted with (grey bars) or without (black bars) addingKDO-N₃.

The following description is the description of an illustrative exampleof click labeling of bacterial membranes of Gram negative bacteria viametabolic modification of the LPS inner-core with modified KDO.

Viable Gram-negative bacteria can specifically incorporate a modifiedKDO into their Lipopolysaccharides, decorating the cell surface with abio orthogonal chemical reporter. Click-chemistry allows furtherlabeling of viable cells, in an overall rapid process as schematicallyshown in FIG. 1.

EXAMPLE 1

assimilation of 8-azido-3,8-dideoxy-D-manno-octulosonic acid (KDO-N₃)and specific detection of Gram negative bacteria

1) Within all potential targets, 3-deoxy-D-manno-octulosonic acid (KDO)appears to be a very attractive candidate. Indeed, KDO is a specific andessential component of the inner core of LPS, [7,8] and has long beenconsidered as being present in the LPS of almost all Gram negativespecies (as well as higher plants and algae), where at least one residueis directly connected to lipid A (FIG. 1A). [9] Due to its vitalimportance, KDO has been considered as a determinant for thecharacterization of Gram negative bacteria, and the KDO pathway as apotential target for the development of new antibacterials. [10] In thispathway (FIG. 1B), arabinose-5-P is condensed with PEP, leading to theformation of KDO-8-P, which is then transformed into free KDO, andfurther activated in the form of the CMP-KDO donor, prior to LPSelaboration.

For all these reasons, it has been sought whether this KDO pathway, as aLPS-specific pathway, could be tolerant enough to incorporate a modifiedKDO, such as 8-azido-8-deoxy-KDO (1, FIG. 2A), into the core of E. coliLPS, and potentially other Gram negative bacteria. Given the presence offree KDO as an intermediate in the pathway, we postulated that if cellpenetration of this analogue of KDO could be sufficient, [11] it couldthen potentially be directly activated, partially replace endogenous KDOinto LPS, and be detected on the cell surface by azide-alkyne clickchemistry (FIG. 2B). [12] Moreover, modification of the C-8 position ofKDO by a bio orthogonal azido group should prevent reverse metabolism byKDO-8-P phosphatase (3.1.3.45), limiting the potential dissemination ofthe chemical reporter into other carbohydrates and metabolites.

Amongst the many potential multi-step synthetic strategies available toaccess 8-azido-3,8-dideoxy-D-manno-octulosonic acid 1, [13] thiscompound has been prepared in a straightforward manner (Scheme 1) [14]adapted from the approach described in 1963 by Ghalambor and Heath fordirect KDO synthesis. [15] Namely 5-azido-5-deoxy-D-arabinofuranose [16](6) was condensed with sodium oxaloacetate (7), leading afterdecarboxylation in slightly acidic conditions to KDO-N₃ (1), which wasisolated as its ammonium salt in 57% yield (86% based on recovered 6).The 5-azido-5-deoxy-D-arabinofuranose precursor 6 could be obtained in avery direct, simple and time-saving strategy from commercialD-arabinose, as described herein after.

1a) Synthesis of ammonium 5-azido-5-deoxy-D-arabinofuranose 6.

The 5-azido-5-deoxy-D-arabinofuranose precursor 6 could be obtained in avery direct, simple and time-saving strategy, avoiding the alternativeuse of a bulky temporary trityl protection: commercial D-arabinose wasdirectly tosylated on the primary position of its furanose forms,further acetylated, and subjected to nucleophilic displacement by azideanion without intermediate purification. At this step, the product couldbe easily separated from any other byproduct by flash chromatography.Final deacetylation afforded 6 in 15% overall yield.

5-azido-5-deoxy-D-arabinofuranose (6)

Commercial D-Arabinose 5 (6.00 g, 40 mmol) was heated at 100° C. for 2hours in pyridine (40 ml). The solution was allowed to cool down,further treated with tosyl chloride (8.38 g, 44 mmol; 1.1 equiv.), andstirred for 16 hours at room temperature. Acetic anhydride (20 ml) wasthen added. After complete acetylation, as determined by TLC, solventswere evaporated, and residual traces were co-evaporated several timeswith toluene. The residue was dissolved in DMF (100 ml), NaN₃ (5.20 g,80 mmol, 2 equiv.) was added, and the suspension was heated at 80° C.for 20 hours. After dilution with ethyl acetate and washing with water,the organic layer was dried over anhydrous magnesium sulfate andconcentrated. The residue was purified by flash chromatography(Petroleum ether/Ethyl acetate 7:3). The first eluted product wasdetermined to be the expected5-azido-1,2,3-tri-O-acetyl-D-arabinofuranose (1.83 g, 15%, α/β˜2:1).LRMS (ESI⁺) 324.0 [M+Na]⁺; HRMS (ESI⁺) calculated for[C₁₁H₁₅N₃NaO₇]+324.0802. found: 324.0802; ¹H NMR (CDCl₃, 360 MHz) δ(ppm): 6.41 (d, 0.33H, J_(1,2) 3.5 Hz, H-1β); 6.23 (d, 0.67H, H-1α);5.40-5.37 (m, 1.34H, H-26, H-36); 5.23 (d, 0.67H, J_(1,2)˜1 Hz, H-2α);5.06 (d, 0.67H, J_(3,4) 4.6 Hz, H-3α); 4.30 (ddd, 0.67H, H-4α);4.16-4.10 (m, 0.33H, H-4β); 3.69 (dd, 0.67H, J_(4,5a) 3.1 Hz, J_(5a,5b)13.5 Hz, H-5aα); 3.61 (dd, 0.33H, J_(4,5b) 3.6, J_(5a,5b) 13.1 Hz,H-5aβ); 3.51-3.43 (m, 1H, H-5bα, H-5bβ); 2.15, 2.13, 2.12, 2.11, 2.11,2.09 (6s, 18H, 6 CH₃CO); ¹³C NMR (CDCl₃, 62.5 MHz) δ (ppm): 170.3,170.0, 169.1 (OC(O)CH₃), 99.2 (C-1α), 93.5 (C-1β), 84.1 (C-4α), 80.8(C-4β), 80.6 (C-3α), 77.4 (C-2α), 75.1 (C-2β), 74.8 (C-3β), 53.0 (C-5β),51.3 (C-5α), 20.9, 20.6, 20.3 (OC(O)CH₃).

1b) Protected 5-azido-1,2,3-tri-O-acetyl-D-arabinose was then dissolvedinto anhydrous methanol (30 ml), treated with a methanolic solution ofMeONa (0.2 mol.I⁻¹, 3 ml) and stirred at room temperature for 3 h underan argon atmosphere. After neutralization (Dowex 50 (H⁺)) filtration,and concentration, 5-azido-5-deoxy-D-arabinofuranose 6 was obtained in99% yield (1.03 g). LRMS (ESI⁺) 198.0 [M+Na]⁺, 40%, 230 [M+MeOH+Na]⁺,100%; HRMS (ESI⁺) calculated for [C₅H₉N₃NaO₄]⁺198.0485. found: 198.0485;¹H NMR (D₂O, 300 MHz) δ (ppm): 5.20 (d, 0.45H, J_(1,2) 3 Hz, H-1β); 5.16(d, 0.55H, J_(1,2) 3 Hz, H-1α); 4.12-4.05 (m, 0.55H, H-4α); 4.02-3.85(m, 2H, H-2α, H-2β, H-3α, H-3β); 3.84-3.76 (m 0.45H, H-4β); 3.56 (dd,0.55H, J_(5a,5b) 13.5, J_(4,5a) 3.0 Hz, H-5aα); 3.51 (dd, 0.45H,J_(5a,5b) 13.0 Hz, J_(4,5a) 3.5 Hz, H-5aβ); 3.35 (dd, 0.55H, J_(4,5b)6.5 Hz, H-5bα); 3.33 (dd, 0.45H, J_(4,5b) 6.5 Hz, H-5bβ); ¹³C NMR (D₂O,75 MHz) δ (ppm): 101.1 C-1α; 95.3 C-1β; 81.4, 81.3, 79.7, 76.4, 75.9,and 74.8 C-2α,2β, 3α, 3β, 4α, 4β; 52.7 C-5β; 51.6, C-5α.

1c) Synthesis of Ammonium 8-azido-3,8-dideoxy-D-manno-octulosonate(1•NH₃).

A cool (4° C.) solution of 5-azido-5-deoxy-D-arabinofuranose 6 (437 mg,2.5 mmol) in water (2.1 mL) was added to a solution of oxaloacetic acid(528 mg, 4.0 mmol) in water (2.5 mL), the pH of which has been adjustedto ˜11 by addition of aqueous NaOH (10M). After being stirred for twohours at room temperature, the solution was neutralized (Dowex 50 (H⁺)),filtrated, and heated 20 min. at 80° C.

After its pH had been adjusted to ˜7 with AcOH (0.5M), the mixture waspurified by anion exchange chromatography (Dowex 1×8 (HCO₂ ⁻)). Initialelution with water gave unreacted 6 (150 mg, 34%). Further elution witha concentration gradient of formic acid (0.5 mol.I⁻¹→2 mol.I⁻¹),freeze-drying, treatment with a Dowex 50 (H⁺) resin, and neutralizationby ammonia (0.2 mol.I⁻¹), gave after concentration, ammonium8-azido-3,8-dideoxy-D-manno-octulosonate (1•NH₃, 400 mg, 57%).

Rf 0.38 (isopropyl alcohol/water 9:1). LRMS (ESI⁻) 262.1 [M-H]⁻, 100%;525.1 [2M-H]⁻, 5%; 547.1 [2M−2H+Na]⁻, 10%; HRMS (ESI⁻) calculated for[C₈H₁₂N₃O₇]⁻262.0681. found: 262.0667. IR ν (cm⁻¹)=3210, 2111 (N₃),1604, 1401, 1077. NMR of 1, like free KDO and derivatives, iscomplicated due to the presence of multiple forms (e.g. α-pyranose (αp,58%), β-pyranose (βp, 4%), α-furanose (αf, 24%), β-furanose (βf, 14%)).Selected NMR data: ¹H NMR (D₂O, 400 MHz) δ (ppm): 3.56 (dd, J_(8a,8b)13.2, J_(7,8a) 2.4 Hz, H-8aαp); 3.39 (dd, J_(7,8b) 6.0 Hz, H-8bαp); 2.55(dd, J_(3a,3b) 14.3 Hz, J_(3a,4) 7.2 Hz, H-3aαf); 2.33 (dd, J_(3a,3b)13.1, J_(3a,4) 6.6 Hz, H-3aβf); 2.26 (dd, J_(3b,4) 7.3 Hz, H-3bβf); 2.03(dd, J_(3b,4) 3.6, H-3bαf); 1.94 (dd, J_(3a,3b)=12.7, J_(3a,4) 12.7 Hz,H-3aαp); 1.84 (dd, J_(3b,4)=4.9 Hz, H-3bαp); 1.71 (dd, J_(3a,3b)J_(3b,4) 12.0 Hz, H-3bβp); ¹³C NMR (D₂O, 100 MHz) δ (ppm): 176.4(C-1αp), 104.1 (C-2αf), 96.2 (C-2αp), 85.3 (C-5αf), 71.5, 68.0, 66.3 and66.0 (C-4-αp, 5αp, 6αp, 7αp), 53.8 (C-8αp), 44.6 (C-3αf), 33.5 (C-3αp).

2) Non-pathogenic E. coli K12, which lacks an O-antigen, [17] wascultured overnight in the presence of KDO-N₃ (1) as described in thefollowing paragraph 3), and further treated, during a time courseexperiment, using optimized copper-catalyzed click conditions [18] asdescribed in the following paragraph 2a), in the presence of aglucose-derived tris-triazolyl ligand (2) [19] and an Alexa Fluor 488fluorophore bearing a terminal alkyne group (3). After 5 min ofincubation, a very bright labeling of bacteria was observed, whilecontrol experiments (in the absence of the KDO-N₃ analogue) did not showany signal (FIG. 3A, B). Fluorescence was carried out as described inthe following paragraph 2b). Fluorescence was mostly evident around thecell periphery suggesting that membrane were preferentially labeled asexpected (FIG. 3C).

2a) Copper Catalyzed Click Chemistry.

Overnight cultures were diluted 1000 times in fresh medium (final volume100 μl) containing KDO-N₃ (4 mM). Bacteria were incubated at 37° C. for12 hours and then washed 3 times with phosphate buffer (0.05 M, pH 7.5)by centrifugation at 13000×g for 1 min at room temperature.

CuSO₄ and TGTA, at a final concentration of 2 mM and 4 mM respectively,were mixed overnight in a phosphate buffer (0.05 M, pH 7.5) at 37° C.under vigorous shaking. Next, aminoguanidine, sodium ascorbate andA488-yne at final concentrations of 4 mM, 5 mM and 0.13 mM respectivelywere added to CuSO₄/TGTA overnight mix. Finally, bacteria werere-suspended in this solution. After 5, 30, 60 or 180 minutes, cellswere washed 3 times with phosphate buffer by centrifugation at 13000×gfor 1 min at room temperature and analyzed by microscopy.

2b) Fluorescence Microscopy.

Bacteria were inoculated onto glass cover slips and then covered with athin (1 mm of thickness) semisolid 1% agar pad made with dilute LB (1/10in phosphate buffer). Images were recorded with epifluorescenceautomated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with aCoolSNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France)and a 100×/1.4 DLL objective. Excitation light was emitted by a 120 Wmetal halide light and signal was monitored using appropriate filters.Digital analysis and image processing were conducted by a customautomation script (Visual Basic) under Metamorph 7.5 (Molecular Devices,Molecular Devices France, France), as previously described [50,51].

3) To further consolidate this approach, its efficiency and specificityhave been tested on three other Gram negative bacteria that use KDO (O86E. coli, Salmonella typhimurium, Legionella pneumophila strain Paris) aswell as three negative controls, including Shewanella oneidensis, whichhas recently been shown to use 8-amino-8-deoxy-KDO instead of KDO [21]and two Gram positive bacteria (Bacillus subtilis, Staphylococcusaureus) which do not produce KDO. [22]

The bacterial strains and growth conditions were as follows. E. coliK12, E. coli 086 and S. typhimurium 12023 were grown in M9 medium(containing also: casamino acid 0.2%, Glucose 0.2%, CaCl₂ 1 mM, MgSO₄ 5mM), S. oneidensis, B. subtilis and S. aureus were grown inLuria-Bertani (LB) medium and L. pneumophila sp. Paris was grown inyeast extract medium supplemented with L-cysteine, ferric pyrophosphateand α-ketoglutarate (YEC). All strains were grown in a rotary shaker(160 rpm) at 37° C. except S. oneidensis which was grown at 28° C.

As expected, when the two E. coli strains, S. typhimurium and L.pneumophila Paris showed efficient and well defined cell-surfacelabeling, and no labeling was observed with S. oneidensis or Grampositive bacteria because these bacteria do not present KDO at theircell surface (FIG. 4 and FIG. 5).

4) Incubation and Reaction on Membrane Filter.

Samples containing bacteria were filtered through 25-mm,0.45-mm-pore-size black polyester membrane filters. Individual membraneswere placed on cellulose pads (25 mm) soaked in 650 μl of nutritivebroth (depending of the bacteria of interest, different nutritive brothcan be used) supplemented with 0.5% pyruvate or catalase (protectagainst oxygen toxic effect) and KDO-N₃ (4 mM) in Petri dishes. Petridishes containing the samples were incubated at 37° C. for a certaintime, (depending the bacteria of interest).

Next, CuSO₄ and TGTA, at a final concentration of 2 mM and 4 mMrespectively, were mixed overnight in a phosphate buffer (0.05 M, pH7.5) at 37° C. under vigorous shaking. Next, aminoguanidine, sodiumascorbate and A488-yne at final concentrations of 4 mM, 5 mM and 0.13 mMrespectively were added to CuSO₄/TGTA overnight mix.

Finally, individual membranes were placed on cellulose pads soaked in650 μl of this solution in Petri dishes and incubated at roomtemperature for 30 minutes.

Images were recorded with epifluorescence automated microscope (NikonTE2000-E-PFS, Nikon, France) equipped with a CoolSNAP HQ 2 camera (RoperScientific, Roper Scientific SARL, France) and a 100×/1.4 DLL objective.Excitation light was emitted by a 120 W metal halide light and signalwas monitored using appropriate filters. Digital analysis and imageprocessing were conducted by a custom automation script (Visual Basic)under Metamorph 7.5 (Molecular Devices, Molecular Devices France,France), as previously described [50,51].

5. In conclusion, it has been demonstrated that the KDO analogue can bemetabolically assimilated and incorporated into the LPS without thenecessity to use genetically modified bacteria. More interestingly thefact that the modified KDO needs first to be metabolically assimilatedled to the use of this method for fast detection (the overall processtaking less than one day) of metabolically active/viable Gram negativebacteria. This last application is very powerful in regard to the factthat detection of viable bacteria needs normally between 2 days and morethan one month depending on the bacterial strain.

Severe pathogens are hiding amongst Gram negative bacteria, and therapid identification of viable cells represents a major sanitarychallenge. The present invention therefore provides a simple strategy tolabel the cell surface of metabolically active Gram negative bacteria,via metabolic incorporation of a modified ulosonic acid or ulosonateresidue such as KDO into their lipopolysaccharide, followed by furtherconjugation using click-chemistry.

Of course, KDO-N₃ assimilation can be subsequently coupled to thefluorescence in situ hybridization (FISH) [24] or any other well-knownprocedure allowing the specific detection of viable bacteria ofinterest.

The ribosomal-RNA (rRNA) approach to microbial evolution and ecology hasbecome an integral part of environmental microbiology. Based on thepatchy conservation of rRNA, oligonucleotide probes can be designed withspecificities that range from the species level to the level of phyla oreven domains. When these probes are labelled for instance withfluorescent dyes or the enzyme horseradish peroxidase, they can be usedto identify single microbial cells directly by fluorescence in situhybridization (FISH) [38]. A new approach has been proposed usingspecifically phage [39].

EXAMPLE 2 Assimilation of8-O-(4-ethynylbenzyl)-3-deoxy-D-manno-octulosonic Acid (KDO-CCH)

This compound 8-O-(4-ethynylbenzyl)-3-deoxy-D-manno-octulosonic acid orits salt (8-O-(4-ethynylbenzyl)-3-deoxy-D-manno-octulosonate), analog ofcompound (Ia-1) can be prepared by the following process:

A cool solution of 5-O-(4-ethynylbenzyl)-D-arabinofuranose in water canbe added to a solution of oxaloacetic acid in water, the pH of whichbeing adjusted to ˜10.5-11 by addition of aqueous NaOH. After beingstirred at room temperature, the solution can be neutralized, andshortly heated at 80° C.

After its pH being adjusted to ˜7 with AcOH, the mixture can purified byanion exchange chromatography on formate resin. After initial elutionwith water, further elution with a concentration gradient of formicacid, freeze-drying, treatment with an acidic resin, neutralization byammonia, and concentration, can give ammonium8-O-(4-ethynylbenzyl)-3-deoxy-D-man no-octulosonate.

The incorporation of the compound in the bacterial LPS and the labelingcan be then carried out with an azido-derived labeling molecule, usingthe same reagents and methods as for KDO-N₃ (example 1).

EXAMPLE 3 Assimilation of9-azido-5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonicAcid and Specific Detection of Legionella Bacteria

This compound9-azido-5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonicacid or its salt(9-azido-5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonate),analog of compound (Ib-1) can be prepared by the following process [52]:

6-azido-2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose can be added to asolution of oxaloacetic acid in water, the pH of which being adjusted to˜10.5-11 by addition of aqueous NaOH, and the mixture being stirred atroom temperature in the presence or not of sodium tetraborate. Furtheradditions of oxaloacetic acid might be necessary to ensure a goodconversion. After neutralization with an acidic resin, filtration andconcentration to a small volume, the solution could be applied to aformate resin, washed with water, and eluted with formic acid. Furtherpurification, for example by reversed-phase HPLC, might be necessary.

The incorporation of the compound in the bacterial LPS and the labelingcan be then carried out with the same reactive groups, the same reagentsand methods as for KDO (example 1).

EXAMPLE 4 Assimilation of5,7-diazidoacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonicAcid and Specific Detection of Legionella Bacteria

This compound5,7-diazidoacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonicacid or its salt(5,7-diazidoacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonate),analog of compound (Ib-1), can be prepared like in the previous example2, by reaction of 2,4-diazidoacetamido-2,4,6-trideoxy-D-mannopyranosewith oxaloacetic acid.

The incorporation of the compound in the bacterial LPS and the labelingthereof can be then carried out as for KDO.

EXAMPLE 5 Use of Various Culture Media

The kind of culture medium is not limitative as both M9 and LB E. coliculture media have been successfully tested.

1) Materials and Methods

1.1) Bacterial Strains and Growth Conditions

E. coli K12 wild type strain grown in M9 medium (containing also:casamino acid 0.2%, glucose 0.2%, CaCl2 1 mM, MgSO4 5 mM) or inLuria-Bertani (LB) medium [54]. Cells were grown in a rotary shaker (200rpm) at 37° C.

1.2) Copper Catalysed Click Chemistry

Overnight cultures were diluted 100 times in fresh medium (final volume200 μl) containing KDO-N3 (1 mM). Bacteria were incubated at 37° C. for12 hours and then washed 3 times with phosphate buffer (0.05 M, pH 7.5)by centrifugation at 14000×g for 2 min at room temperature.

A click pre-mix composed of CuSO4 and TGTA, at a final concentration of2 mM and 4 mM respectively, is incubated overnight in a phosphate buffer(0.05 M, pH 7.5) at 37° C. under vigorous shaking. Next, aminoguanidine, sodium ascorbate and A488-yne at final concentrations of 4mM, 5 mM and 0.13 mM respectively were added to the overnight clickpre-mix. Bacteria were then re-suspended in this solution and incubated30 minutes at 37° C. under shaking. Finally, cells were washed 3 timeswith phosphate buffer by centrifugation at 14000× g for 2 min at roomtemperature.

1.3) Fluorescence Microscopy

Microscopic analysis were performed after Copper click chemistry onbacterial inoculum placed onto glass cover slips and covered with a thin(1 mm thick) semisolid 1% agar pad made with dilute LB (1/10 inphosphate buffer). Images were recorded with an epifluorescenceautomated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with aCool SNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France)and a 100×/1.4 DLL objective. Excitation light was emitted by a 120 Wmetal halide light and signal was monitored using appropriate filters.Digital analysis and image processing were conducted by a customautomation script (Visual Basic) under Metamorph 7.5 (Molecular Devices,Molecular Devices France, France).

2) Results

KDO assimilation occurs whatever the culture medium used.

Analysis has been performed on 616 E. coli cells from LB medium and 598E. coli cells from minimum medium (M9).

A higher KDO assimilation is obtained when cells were grown in LB mediumcompared to M9 medium. However, both media are convenient and sufficientto detect and quantify cultivable bacteria.

EXAMPLE 6 Detection and Counting of Cultivable/Living and Dead E. coliCells

Known numbers of both cultivable E. coli capable of multiplying and deadE. coli have been contacted with KDO-N3. KDO-N3 was incorporated only inliving E. coli. KDO-N3 was incorporated in all of the living E. coli.

1) Materials and Methods

1.1) Bacterial Strains and Growth Conditions

E. coli K12 was grown in Luria-Bertani (LB) medium in a rotary shaker(200 rpm) at 37° C. Dead E. coli—K12 were obtained after heating at 120°C. for 15 minutes.

1.2) Copper Catalysed Click Chemistry (as Disclosed in Example 5).

1.3) Bacterial Counting and Fluorescence Microscopy

1.3.1) For total bacterial scoring, cells were fixed and stained in asolution of paraformaldehyde 3%—DAPI 2 μg/ml and filtered on an isoporepolycarbonate membrane (Milipore).

1.3.2) Colony forming units (CFU) were also monitored by platingdilutions before Copper click chemistry.

1.3.3) Microscopic analyses were performed after Copper click chemistryon bacterial inoculum placed onto glass cover slips and covered with athin (1 mm thick) semisolid 1% agar pad made with dilute LB (1/10 inphosphate buffer). Images were recorded with an epifluorescenceautomated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with aCool SNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France)and a 100×/1.4 DLL objective. Excitation light was emitted by a 120 Wmetal halide light and signal was monitored using appropriate filters.Digital analysis and image processing were conducted by a customautomation script (Visual Basic) under Metamorph 7.5 (Molecular Devices,Molecular Devices France, France).

2) Results

2.1) the total cell concentration calculated by the total scoringdisclosed in 1.3.1) was 9 10⁹+/−0.1 10⁹ cells/ml while the cultivablecell concentration counted by the colonies counting disclosed in 1.3.2)was 4 10⁹+/−0.15 10⁹ cfu/ml. These result indicates that 44.5% (+/−4%)of all cells present in this sample are cultivable.

2.2) the counting by fluorescence after click chemistry as disclosed inparagraphs 1.2/1.3.3) provided the following results. One Sample wasincubated without KDO-N3, and one sample was incubated with KDO-N3.Fluorescence analysis has been performed on 1878 E. coli cells withKDO-N3 and 3039 E. Coli cells without KDO-N3. It enabled to determine athreshold of arbitrary fluorescence unit of 30 below which the cells aredead and above which the cells are cultivable. Next, applying thisthreshold (30), it was possible to count cell identified as dead (below30) and identified as cultivable (above 30) on bacteria having reactedthrough click chemistry as in 1.3.3) Doing this evaluation, 1587 deadcells (KDO-N3−) and 1452 cultivable cells (KDO-N3+) leading to a 47.7%of cultivable cell were found. This value appears statisticallyidentical to the one obtained using dead and cultivable cells, 44.5%demonstrating that only cultivable cells are detected by the method ofthe present invention.

EXAMPLE 7 Detection of Only Gram-Cultivable Cells

A mixture of Gram+ bacteria (B. subtilis) and Gram− bacteria (E. Coli)have been contacted with KDO-N3. Only cultivable Gram− bacteria waslabeled with KDO-N3 and detected.

1) Material and Method

1.1) Bacterial Strains and Growth Conditions

E. coli K12 wild type strain rendered fluorescent via m-cherry

[55], E. coli K12 sodA-mCherry [55] and B. subtilis were grown inLuria-Bertani (LB) medium in a rotary shaker (200 rpm) at 37° C.

1.2) Copper Catalysed Click Chemistry (as Disclosed in Example 6)

1.3) Bacterial Counting and Fluorescence Microscopy (as Disclosed inExample 5)

2) Results

B. subtilis and E. coli were grown in LB medium during 12 hours. Afterthat time cultivable cell concentration from E. coli (1.66 10⁹ cfu/ml)and B. subtilis (5.9 10⁸ cfu/ml) have been identified by countingdisclosed in paragraph 1.32), indicating that 26% of the cultivablecells were B. subtilis and 74% were E. coli.

Moreover, because an E. coli sodA-mCherry strain was used, it waspossible to differentiate E. coli and B. subtilis using fluorescence asdisclosed above but before click chemistry. Among the 696 analyzedcells, 170 were mCherry negative (24.4%) representing by consequence B.subtilis, and 526 mCherry positive (75.6%) representing by consequenceE. coli. These results are confirmed with the percentages obtained fromthe cfu values.

Then, all of the bacteria of the same sample have been subjected to theabove click chemistry reaction and the results of counting viafluorescence are given in the following table 1.

Within the KDO-N3 negative cells, the number of mCherry negative cellsrepresenting by consequence B. subtilis (166) and the number of mCherrypositive cells representing by consequence the number of E. coli cells(9) were evaluated. Within the KDO-N3 positive cells, the number ofmCherry negative cells representing by consequence the number of B.subtilis (4) and the number of mCherry positive cells representing byconsequence the number of cultivable E. coli (517) have been evaluated.

Using all these values, it was possible to identify 4 B. subtilis asfalse positive (about 2%). By contrasts the 9 E. coli KDO negative(about 2%) can be either false positive or false negative since thestandard method error range is about 10%.

KDO- KDO- N3+ N3− Total mCherry + (E. coli) 517 9 526 mCherry − (B.subtilis) 4 166 170 Total 521 175 696

These above results demonstrate therefore that KDO-N3 has well beenassimilated in substantially only cultivable E. coli cells and not incultivable B. subtilis cells.

EXAMPLE 8 Detection Via Biotin-Alkyne

After Kdo-N3 assimilation, click chemistry has been performed usingbiotin-alkyn and viable E. coli cells were detected using an anti-biotinantibody coupled to fluorochome Alexa Fluor A494. As observed bycomparing cultivable counting and Kdo positive cell counting, all viableE. coli bacteria were detected by the following experimental procedure.

1) Bacterial Strains and Growth Conditions

E. coli K12 wild type strain is grown in Luria-Bertani (LB) medium in arotary shaker (200 rpm) at 37° C.

2) Copper Catalyzed Click Chemistry:

Overnight cultures were diluted 100 times in fresh medium (final volume200 μl) containing KDO-N3 (1 mM). Bacteria were incubated at 37° C. for12 hours, washed 3 times with phosphate buffer (0.05 M, pH 7.5) bycentrifugation at 14000×g for 2 min at room temperature. A click pre-mixcomposed of CuSO4 and TGTA, at a final concentration of 2 mM and 4 mMrespectively, was incubated overnight in phosphate buffer at 37° C.under vigorous shaking. Next, aminoguanidine, sodium ascorbate at finalconcentrations of 4 mM and 5 mM respectively were added to the overnightpre-mix click. This “click-mix” was added to the washed culture andsupplemented or not with biotin-alkyn (Carbosynth) (1 to 100 μg) andincubated for 1 to 60 min at 37° C. under shaking.

3) Bacterial Counting and Fluorescence Microscopy:

For total bacterial scoring both before and after the Copper clickchemistry, cells were fixed and stained in a solution ofparaformaldehyde 3%—DAPI 2 μg/ml and filtered on an isoporepolycarbonate membrane (Milipore).

An anti-biotin antibody coupled to Alexa Fluor A594 (Jackson ImmunoResearch) was used diluted 10 to 1000 times to label bacteria. Colonyforming units (CFU) were also monitored by plating dilutions beforeCopper click chemistry.

Microscopic analyses were performed after Copper click chemistry onbacterial inoculum placed onto glass cover slips and covered with a thin(1 mm thick) semisolid 1% agar pad made with dilute LB (1/10 inphosphate buffer). Images were recorded with an epifluorescenceautomated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with aCoolSNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France)and a 100×/1.4 DLL objective. Excitation light was emitted by a 120 Wmetal halide light and signal was monitored using appropriate filters.Digital analysis and image processing were conducted by a customautomation script (Visual Basic) under Metamorph 7.5 (Molecular Devices,Molecular Devices France, France).

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1. A method for labeling specifically living bacteria of a givencategory of bacteria in a sample comprising bacteria, the methodcomprising the steps of: a) incubating said bacteria of said sample withat least one analog of a monosaccharide compound, said monosaccharidebeing an endogenous monosaccharide residue of glycans of the outermembrane of such given category of bacteria, the said endogenousmonosaccharide residue comprising an ulosonic acid or ulosonate saltresidue, the said analog of a monosaccharide compound being a modifiedmonosaccharide substituted at a given position by a first reactivechemical group capable to react with a second reactive group of alabeling molecule, the said given position being preferably a positionwhich comprises a free group in the said endogenous monosaccharideresidue incorporated within said glycans of the outer membrane of thebacteria, b) contacting said bacteria with a said labeling moleculecomprising a said second reactive group, for generating the reaction ofsaid first reactive group of said analog residue incorporated withinsaid glycans of the outer membrane of said living bacteria with saidsecond reactive group of said labeling molecule.
 2. A method accordingto claim 1 for labeling specifically bacteria capable of multiplyingwherein said bacteria are incubated in a culture medium in or on whichsaid bacteria are capable to multiply.
 3. A method according to claim 1,comprising the further step of: c) detecting living bacteria indetecting whether said bacteria comprise said labeling molecule bound tothe glycans of their outer membrane and/or immobilizing said livingbacteria bearing said labeling molecule onto a solid substrate.
 4. Amethod according to claim 3 wherein said labeling molecule is adetectable molecule comprising a detectable substance or capable toreact or to be bound to a detectable substance or said labeling moleculeis a first molecule bearing a said second reactive group, said firstmolecule being capable to react or to be bound to a second moleculeand/or to a solid substrate, preferably said second molecule comprisinga detectable substance and/or said second molecule being bound to a saidsolid substrate.
 5. A method according to claim 4 for specificallydetecting living bacteria of a given category of bacteria in a samplecomprising bacteria, wherein said labeling molecule is a detectablemolecule comprising a detectable substance, the method comprising thestep c) of detecting living bacteria in detecting whether said bacteriacomprise said detectable molecule bound to the glycans of their outermembrane.
 6. A method according to claim 4 wherein said labelingmolecule is a first ligand or first binding protein bearing a saidsecond reactive group and in step c) said living bacteria coupled tosaid first ligand or first binding protein is detected and/orimmobilized by contacting said first ligand or first binding proteinwith a second ligand or second binding protein reacting or bindingspecifically to said first ligand or first binding protein.
 7. A methodaccording to claim 6 wherein said labeling molecule is a first ligand,preferably biotin, bearing a said second reactive group, and in step c)said living bacteria coupled to said first ligand are detected byreaction of said bacteria with an antibody specific to said firstligand, said antibody bearing a detectable substance, preferably afluorochrome or luminescent molecule or an enzyme.
 8. A method accordingto claim 4 wherein the said detectable substance is a fluorochrome orluminescent molecule detectable by fluorescence or luminescence.
 9. Amethod according to claim 1 wherein the said analog of monosaccharidecompound is an ulosonic acid having one of the following formulas (I) or(II), or an ulosonate salt thereof:

Wherein A, B and C can be independently H, OH, NH₂, OH and NH₂ beingsubstituted or not by protecting groups thereof, preferably substitutedby alkyl, hydroxyalkyl, acyl, formyl or imidoyl groups, and D is analkyl chain in C₂ to C₄, each carbon being substituted or not by OH orNH₂ substituted or not by protecting groups thereof, preferably byalkyl, hydroxyalkyl, acyl, formyl or imidoyl groups, and at least one ofA, B, C or D groups is substituted by a said first reactive group.
 10. Amethod according to claim 1 wherein the said analog of monosaccharide isa substituted octulosonic acid or octulosonate salt compound or asubstituted nonulosonic acid or nonulosonate salt compound.
 11. A methodaccording to claim 10 for detecting specifically living Gram negativebacteria wherein the said given category of bacteria is the category ofthe Gram negative bacteria and said endogenous monosaccharide residue ofsaid LPS layer of the outer membrane of the bacteria is adeoxyoctulosonic acid or deoxyoctulosonate residue, and said analog ofmonosaccharide compound is a substituted deoxyoctulosonic acid ordeoxyoctulosonate compound.
 12. A method according to claim 11 whereinthe said analog of deoxyoctulosonic acid or deoxyoctulosonate compoundis substituted by a said reactive group at one position selected amongthe positions 3, 4, 5, 7 and 8 of the monosaccharide, preferably 3, 7and
 8. 13. A method according to claim 9 wherein the said analog ofdeoxyoctulosonic acid or deoxyoctulosonate compound of formula (I) or(II) is substituted by a said first reactive group R₁ at the position 8wherein D=—CHOH—CH₂—R₁, A=H, B=OH, C=OH in formula (I) orD=—CHOH—CHOH—CH₂—R₁, A=H, B=OH in formula (II).
 14. A method accordingto claim 11 wherein the said Gram negative bacteria comprise E. coli,Salmonella typhimurium, Legionella pneumophila and Pseudomonasaeruginosa.
 15. A method according to claim 1 for labeling specificallyliving Legionella pneumophila bacteria and the said given category ofbacteria is the category of the Legionella pneumophila bacteria and saidendogenous monosaccharide residue of said LPS layer of the outermembrane of the bacteria is a 4-epilegionaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid) or4-epilegionaminate residue, or a legionaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid)or legionaminate residue, and the said analog of a monosaccharidecompound is respectively a substituted 4-epilegionaminic acid or4-epilegionaminate compound, or a substituted legionaminic acid orlegionaminate compound, preferably substituted at one position selectedamong the positions 3, 4, 5, 7, 8 and 9 of the monosaccharide cycle,preferably 5, 7 and
 9. 16. A method according to claim 1 for labeling,preferably detecting, specifically living Pseudomonas aeruginosabacteria and the said given category of bacteria is the category of thePseudomonas aeruginosa bacteria and said endogenous monosaccharideresidue of said LPS layer of the outer membrane of the bacteria is a8-epilegionaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid)or 8-epilegionaminate residue, or a pseudaminic acid(5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid)or pseudaminate residue, and the said analog of a monosaccharidecompound is respectively a substituted 8-epilegionaminic acid or8-epilegionaminate compound, or a substituted pseudaminic acid orpseudaminate compound, preferably substituted at one position selectedamong the positions 3, 4, 5, 7, 8 and 9 of the monosaccharide cycle,preferably 5, 7 and
 9. 17. A method according to claim 1 wherein thesaid first reactive group is selected among groups consisting in orbearing the group azido and groups consisting in or bearing the groupalkyne, and the said second reactive group is selected among groupsconsisting in or bearing respectively the groups alkyne and azido, andreacting the said azido reactive group with the said alkyne reactivegroup is carried out in performing an azide alkyne cycloaddition.
 18. Amethod according to claim 17 wherein the reaction is carried out incopper catalyzed conditions in the presence of a tris-triazolyl ligand,preferably TGTA.
 19. A method according to claim 2 for numbering livingbacteria wherein the said incubation of step a) and reaction of step b)are carried out on a solid substrate, preferably a membrane filter sothat the cultivated bacteria emanating from a same original bacteriumwhich has been multiplied are grouped together and can be visualizedwith a microscope and the said detectable molecule can be detected byvisualization with a said microscope.
 20. A kit for carrying out themethod of claim 1 comprising: a said analog of a monosaccharide compoundcomprising an ulosonic acid or ulosonate compound substituted at a givenposition by a said first reactive chemical group, and a said labelingmolecule comprising a said second reactive group capable of reactingwith said first reactive group, and reactants for generating thereaction of said first reactive group of said analog residueincorporated within said glycans of the outer membrane of said bacteriawith said second reactive group of said labeling molecule, and a cultureor incubation medium allowing the growth of a said given category ofbacteria, preferably specific to the growth of said given category ofbacteria.
 21. A kit according to claim 20 for carrying out the method ofclaim 19 further comprising: a said detectable molecule comprising asaid second reactive group capable of reacting with said first reactivegroup, and a solid medium allowing visualization of the bacteria afterincubating with the said analog of a monosaccharide compound, saidreactants and said detectable molecule.
 22. A kit according to claim 20comprising: a said analog of a monosaccharide compound being adeoxyoctulosonic acid or deoxyoctulosonate salt compound substituted bya said first reactive group comprising an azido or alkyne group, and asaid second reactive group of the detectable molecule bearing an alkyneor, respectively, azido group, and said reactants comprising a coppercatalyst and a tris-triazolyl ligand.